STABILIZATION OF YEAST ARTIFICIAL CHROMOSOME CLONES IN A RAD54-3 RECOMBINATION-DEFICIENT HOST STRAIN

The cloning and propagation of large fragments of DNA on yeast artific ial chromosomes (YACs) has become a routine and valuable technique in genome analysis, Unfortunately, many YAC clones have been found to und ergo rearrangements or deletions during the cloning process. The frequ ency of transformation-associated alterations and mitotic instability can be reduced in a homologous recombination-deficient yeast host stra in such as a rad52 mutant, RAD52 is one member of an epistatic group o f genes required for the recombinational repair of double-strand break s in DNA, rad52 mutants grow more slowly and transform less efficientl y than RAD(+) strains and are therefore not ideal hosts for YAC librar y construction, We have investigated the ability of both null and temp erature-sensitive alleles of RAD54, another member of the RAD52 epista sis group, to prevent rearrangements of human YAC clones containing ta ndemly repeated DNA sequences. Our results show that the temperature-s ensitive rad54-3 allele blocks mitotic recombination between tandemly repeated DYZ3 satellite sequences and significantly stabilizes a human DYZ5 satellite-containing YAC clone. Yeast carrying the rad54-3 mutat ion can undergo meiosis, have growth and transformation rates comparab le with RAD(+) strains, and therefore represent improved YAC cloning h osts.